71 research outputs found
An open environment CT-US fusion for tissue segmentation during interventional guidance.
Therapeutic ultrasound (US) can be noninvasively focused to activate drugs, ablate tumors and deliver drugs beyond the blood brain barrier. However, well-controlled guidance of US therapy requires fusion with a navigational modality, such as magnetic resonance imaging (MRI) or X-ray computed tomography (CT). Here, we developed and validated tissue characterization using a fusion between US and CT. The performance of the CT/US fusion was quantified by the calibration error, target registration error and fiducial registration error. Met-1 tumors in the fat pads of 12 female FVB mice provided a model of developing breast cancer with which to evaluate CT-based tissue segmentation. Hounsfield units (HU) within the tumor and surrounding fat pad were quantified, validated with histology and segmented for parametric analysis (fat: -300 to 0 HU, protein-rich: 1 to 300 HU, and bone: HU>300). Our open source CT/US fusion system differentiated soft tissue, bone and fat with a spatial accuracy of ∼1 mm. Region of interest (ROI) analysis of the tumor and surrounding fat pad using a 1 mm(2) ROI resulted in mean HU of 68±44 within the tumor and -97±52 within the fat pad adjacent to the tumor (p<0.005). The tumor area measured by CT and histology was correlated (r(2) = 0.92), while the area designated as fat decreased with increasing tumor size (r(2) = 0.51). Analysis of CT and histology images of the tumor and surrounding fat pad revealed an average percentage of fat of 65.3% vs. 75.2%, 36.5% vs. 48.4%, and 31.6% vs. 38.5% for tumors <75 mm(3), 75-150 mm(3) and >150 mm(3), respectively. Further, CT mapped bone-soft tissue interfaces near the acoustic beam during real-time imaging. Combined CT/US is a feasible method for guiding interventions by tracking the acoustic focus within a pre-acquired CT image volume and characterizing tissues proximal to and surrounding the acoustic focus
Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation.
Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response
Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis
Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.publishedVersio
Magnetic Resonance Thermometry at 7T for Real-Time Monitoring and Correction of Ultrasound Induced Mild Hyperthermia
While Magnetic Resonance Thermometry (MRT) has been extensively utilized for non-invasive temperature measurement, there is limited data on the use of high field (≥7T) scanners for this purpose. MR-guided Focused Ultrasound (MRgFUS) is a promising non-invasive method for localized hyperthermia and drug delivery. MRT based on the temperature sensitivity of the proton resonance frequency (PRF) has been implemented in both a tissue phantom and in vivo in a mouse Met-1 tumor model, using partial parallel imaging (PPI) to speed acquisition. An MRgFUS system capable of delivering a controlled 3D acoustic dose during real time MRT with proportional, integral, and derivative (PID) feedback control was developed and validated. Real-time MRT was validated in a tofu phantom with fluoroptic temperature measurements, and acoustic heating simulations were in good agreement with MR temperature maps. In an in vivo Met-1 mouse tumor, the real-time PID feedback control is capable of maintaining the desired temperature with high accuracy. We found that real time MR control of hyperthermia is feasible at high field, and k-space based PPI techniques may be implemented for increasing temporal resolution while maintaining temperature accuracy on the order of 1°C
Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI.
Manual palpation is a common and very informative diagnostic tool based on estimation of changes in the stiffness of tissues that result from pathology. In the case of a small lesion or a lesion that is located deep within the body, it is difficult for changes in mechanical properties of tissue to be detected or evaluated via palpation. Furthermore, palpation is non-quantitative and cannot be used to localize the lesion. Magnetic Resonance-guided Focused Ultrasound (MRgFUS) can also be used to evaluate the properties of biological tissues non-invasively. In this study, an MRgFUS system combines high field (7T) MR and 3 MHz focused ultrasound to provide high resolution MR imaging and a small ultrasonic interrogation region (~0.5 x 0.5 x 2 mm), as compared with current clinical systems. MR-Acoustic Radiation Force Imaging (MR-ARFI) provides a reliable and efficient method for beam localization by detecting micron-scale displacements induced by ultrasound mechanical forces. The first aim of this study is to develop a sequence that can concurrently quantify acoustic radiation force displacements and image the resulting transient shear wave. Our motivation in combining these two measurements is to develop a technique that can rapidly provide both ARFI and shear wave velocity estimation data, making it suitable for use in interventional radiology. Secondly, we validate this sequence in vivo by estimating the displacement before and after high intensity focused ultrasound (HIFU) ablation, and we validate the shear wave velocity in vitro using tissue-mimicking gelatin and tofu phantoms. Such rapid acquisitions are especially useful in interventional radiology applications where minimizing scan time is highly desirable
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In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles
Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies
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Unimicellar hyperstars as multi-antigen cancer nanovaccines displaying clustered epitopes of immunostimulating peptides
Unimicellar hyperstar macromolecular chimeras displaying multiple melanoma peptide antigens were prepared primarily via a combination of click chemistry and esterification reactions starting from a biodegradable hyperbranched polymer template. Solubilization of the hyperstars in aqueous solution afforded a multi-antigen unimicellar cancer nanovaccine of about 20 nm. The nanovaccine showed good biocompatibility and uptake by dendritic cells in vitro. An in vivo evaluation of the nanovaccine therapeutic efficacy against melanoma in mice implanted with B16OVA tumors revealed significantly greater T-cell recruitment and improved survival rates for mice treated with nanovaccine and adjuvant compared to non-treated mice
Shear wave visualization.
<p>(A) Effect of the second Motion Encoding Gradient (MEG) on the phase images. TE = 23.8 ms, δ = 7 ms, G<sub>e</sub> = 140 mT/m. The images are 85 × 85 mm<sup>2</sup> in size. MR sequences (i,ii) and (iii,iv) the corresponding phase images of the shear wave. (B) Shear wave velocity estimation. (i). MR sequence used to estimate shear wave speed by varying delay t. (ii). Shear profiles analysis. Lines are extended from the focus outwards in all directions (360 degree sweep, at θ = 1 degree increment) to determine the distance of the peak minima from the focus. Propagation speed of the shear wave was calculated by measuring the radii of the peak and minima as a function of the delay time. (C) Shear wave propagation in 8% gelatin phantom. Phase images at shear wave propagation times of 10, 12, 14 and 18 ms corresponding to time delays t = 0, 1, 2 and 4 ms. (D) Distance of the peak minima from the focus versus delay time t in 8% gelatin and tofu phantoms. (E) Shear wave velocity in 8, 10% gelatin and tofu phantoms. (F) Shear wave velocity map.</p
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Magnetic resonance imaging assessment of effective ablated volume following high intensity focused ultrasound.
Under magnetic resonance (MR) guidance, high intensity focused ultrasound (HIFU) is capable of precise and accurate delivery of thermal dose to tissues. Given the excellent soft tissue imaging capabilities of MRI, but the lack of data on the correlation of MRI findings to histology following HIFU, we sought to examine tumor response to HIFU ablation to determine whether there was a correlation between histological findings and common MR imaging protocols in the assessment of the extent of thermal damage. Female FVB mice (n = 34), bearing bilateral neu deletion tumors, were unilaterally insonated under MR guidance, with the contralateral tumor as a control. Between one and five spots (focal size 0.5 × 0.5 × 2.5 mm3) were insonated per tumor with each spot receiving approximately 74.2 J of acoustic energy over a period of 7 seconds. Animals were then imaged on a 7T MR scanner with several protocols. T1 weighted images (with and without gadolinium contrast) were collected in addition to a series of T2 weighted and diffusion weighted images (for later reconstruction into T2 and apparent diffusion coefficient maps), immediately following ablation and at 6, 24, and 48 hours post treatment. Animals were sacrificed at each time point and both insonated/treated and contralateral tumors removed and stained for NADH-diaphorase, caspase 3, or with hematoxylin and eosin (H&E). We found the area of non-enhancement on contrast enhanced T1 weighted imaging immediately post ablation correlated with the region of tissue receiving a thermal dose CEM43 ≥ 240 min. Moreover, while both tumor T2 and apparent diffusion coefficient values changed from pre-ablation values, contrast enhanced T1 weighted images appeared to be more senstive to changes in tissue viability following HIFU ablation
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